Higher aliphatic 2,4-diketones: a ubiquitous lipid class with chelating properties, in search of a physiological function

A group of homologous 2,4-diketones with chain lengths from C13 to C25, derived from fatty acids, is present in human and mammalian tissues and urine. The presence of this lipid class, members of which possess marked chelating properties, adequately accounts for the well-documented antiallergic activity of tissue and urine extracts on isolated smooth muscle preparations and in guinea pigs in vivo. This review summarizes the investigations leading to the discovery of this lipid class. It is postulated that the observed antiallergic activity of 2,4-diketones may be explained by the intracellular sequestration of calcium ions transiently liberated from intracellular storage pools after an agonist-receptor interaction, thus blocking response to the agonist. The true physiological function (or functions) of this lipid class has not been defined. The chelating properties of 2,4-diketones suggest their involvement in intracellular ionic interactions.     

Purification and properties of two oxidoreductases catalyzing the enantioselective reduction of diacetyl and other diketones from baker's yeast

The NADPH-linked diacetyl reductase system from the cytosolic fraction of Saccharomyces cerevisiae has been resolved into two oxidoreductases catalyzing irreversibly the enantioselective reduction of diacetyl (2,3-butanedione) to (S)- and (R)-acetoin (3-hydroxy-2-butanone) [so-called (S)- and (R)-diacetyl reductases] (EC 1.1.1.5) which have been isolated to apparent electrophoretical purity. The clean-up procedures comprising streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B column chromatography, affinity chromatography on Matrex Gel Red A and Superose 6 prep grade filtration led to 120-fold and 368-fold purifications, respectively. The relative molecular mass of the (R)-diacetyl reductase, estimated by means of HPLC filtration on Zorbax GF 250 and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was 36,000. The (R)-enzyme was most active at pH 6.4 and accepted in addition to diacetyl C5-, C6-2,3-diketones, 1,2-cyclohexanedione, 2-oxo aldehydes and short-chain 2- and 3-oxo esters as substrates. The enzyme was characterized by high enantioselectivity and regiospecificity. The Km values for diacetyl and 2,3-pentanedione were determined as 2.0 mM. The Mr of the (S)-diacetyl reductase was determined as 75,000 by means of HPLC filtration of Zorbax GF 250. The enzyme decomposed into subunits of Mr 48,000 and 24,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The optimum pH was 6.9. The purified (S)-enzyme reduced stereospecifically a broad spectrum of substrates, comprising 2,3-, 2,4- and 2,5-diketones, 2-oxo aldehydes, 1,2-cyclohexanedione and methyl ketones as well as 3-, 4- and 5-oxo esters. The 2,3- and 2,4-diketones are transformed to the corresponding (S)-2-hydroxy ketones; 2,5-hexanedione, however, was reduced to (S,S)-2,5-hexanediol. The Km values for diacetyl and 2,3-pentanedione were estimated as 2.3 and 1.5 mM, respectively. Further characterization of the (S)-diacetyl reductase revealed that it is identical with the so-called '(S)-enzyme', involved in the enantioselective reduction of 3-, 4- and 5-oxo esters in baker's yeast.     

Chemical Composition and Recrystallization of Epicuticular Waxes: Coiled Rodlets and Tubules

Abstract: Coiled rodlets characterize several non-related taxa within the angiosperms. They often occur together with tubules but sometimes also with platelets or transitional forms between them. The ultrastructure chemistry, and recrystallization of epicuticular waxes of three species were investigated by high-resolution scanning electron microscopy, gas chromatography, and mass spectrometry. Whereas Buxus sempervirens (Buxaceae) and Chrysanthemum segetum (Asteraceae) show coiled rodlets in combination with tubules, Leymus arenarius (Poaceae) exhibits tubules but no coiled rodlets. Chemical analyses reveal that the predominating β-diketones of all species differ completely in their molecular structure. Those of the former two species are mainly substituted in carbon atom positions up to 12. In contrast, the wax of L. arenarius contains only hentriacontane-14,16-dione and 25-hydroxy-hentriacontane-14,16-dione. Standard solutions of the total waxes from B. sempervirens, C. segetum and L. arenarius, the purified β-diketone fraction from C. segetum and hentriacontane-14,16-dione from Secale cereale were taken for recrystallization experiments under different conditions in relation to solvent and crystallization velocity. It was demonstrated that coiled rodlets grew exclusively from total waxes of B. sempervirens and C. segetum, and its β-diketone fraction but never from L. arenarius wax or pure hentriacontane-14,16-dione. The recrystallization experiments pointed out that conditions, such as the chemical environment and physical factors, strongly influence the formation of coiled rodlets and tubules. It is concluded that coiled rodlets are formed by self-assembly in close dependence on the position of β-diketo substitution. The future role of β-diketones in the classification of coiled rodlets within wax crystals is discussed.     

Analysis of a range of crown gall and normal plant tissues for Ti plasmid-determined compounds

Summary Twenty-five plant tissues from several species, including thirteen crown gall tissues, were analysed for the full range of unusual compounds (the opines) whose synthesis in crown gall cells and utilization by Agrobacterium tumefaciens are genetically determined by the Ti plasmids found in this bacterial species. A technique for the analysis of the non-guanidino opines by GC and GC/MS is described. None of the opines were detected in any of the various normal tissues analysed. In the crown gall tissues, on the other hand, these compounds were often present at very high levels. The type of opines found in the crown gall tissues was dependent on the strain of initiating bacterium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GC gas chromatography - GC/MS coupled gas chromatography-mass spectrometry - HFB heptafluorobutyryl - SIM selected ion monitoring - TLC thin layer chromatography     

Specific determination of threonine in biological samples by gas chromatography with electron capture detection

Threonine was oxidized into acetaldehyde at 0 degrees C for 30 min with periodic acid. The acetaldehyde formed was converted to a hydrazone with 2,4-dinitrophenyhydrazine. The hydrazone was extracted with n-heptane and quantified by gas liquid chromatography with electron capture detection. An internal standard, 2-amino-3-hydroxyhexanoic acid, was used. The calibration curve of threonine was linear up to 200 nmol in 200 microl sample solution and the determination limit of threonine was 1 nmol in 200 microl sample solution. The recoveries were 100.0, 94.0 and 100.0% from homogenates of octopus tentacles and blood plasma and rat livers, respectively. This method was applied to the determination of threonine in tissues of rats given threonine and starved octopuses. This threonine determination method has been used for studies on the metabolism of d-lactate.     

Effect of 2,4-dichlorophenoxyacetic acid on the activity of o-methyltransferase in carrot cell culture

The activity of caffeic acid-O-methyltransferase (OMT) in carrot cells was greatly affected by the amount of 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented to the culture medium. The OMT fraction was purified by (NH₄)₂SO₄ followed by ultrafiltration and gel filtration or DEAE-Sephadex chromatography after cells were cultured in the medium containing [2-¹⁴C]-2,4-D. Thus, this purified fraction revealed high OMT activity and was still radioactive. The OMT activity was about eight-fold higher (or more) in cells cultured at 0.05 ppm 2,4-D than in those at 1.0 ppm 2,4-D. The ratio of radioactivity to OMT activity was about four-fold higher in cells cultured at 1.0 ppm 2,4-D than those at 0.05 ppm 2,4-D. On the other hand, the OMT fraction was separated into two radioactive protein fractions by DEAE-Sephadex chromatography. The radioactive fractions became Et₂O-soluble after HCl hydrolysis, but not after salt-urea treatment. From these results, it was concluded that 2,4-D is covalently bound to proteins in the OMT fraction. Such 2,4-D protein conjugates may play a role in the regulation of OMT activity.     

Detection of 4-hydroxynonenal and other lipid peroxidation products in the liver of bromobenzene-poisoned mice

Lipid peroxidation in cellular membranes leads to the formation of toxic aldehydes. One product provided with particular reactivity has been identified as 4-hydroxynonenal and thoroughly studied as one of the possible mediators of the cellular injury induced by pro-oxidants. In the present study we have searched for the presence of 4-hydroxynonenal and other lipid peroxidation products in the liver of bromobenzene-poisoned mice, since under this experimental condition the level of lipid peroxidation is much greater than in the case of CCl4 or BrCCl3 hepatotoxicity. 4-Hydroxynonenal was looked for in liver extracts as either free aldehyde or its 2,4-dinitrophenylhydrazone derivative. In both cases, by means of thin-layer chromatography (TLC) and high-pressure liquid chromatography, a well resolved peak corresponding to the respective standards (free aldehyde or 2,4-dinitrophenylhydrazone derivative) was obtained. Total carbonyls present in the liver of intoxicated animals were detected as 2,4-dinitrophenylhydrazone derivatives. The hydrazones were pre-separated by TLC into three fractions according to different polarity (polar, non-polar, fraction I, and non-polar, fraction II). The amounts of carbonyls present in each fraction were determined by ultraviolet-visible spectroscopy. 'Non-polar carbonyls, fraction II' were further fractionated by TLC. The fraction containing alkanals and alk-2-enals was analyzed by high-pressure liquid chromatography and several aldehydes were identified. In addition, protein bound carbonyls were determined in the liver of bromobenzene-treated mice. The biological implications of the finding of 4-hydroxynonenal and other carbonyls in vivo in an experimental model of hepatotoxicity are discussed.     

Protein kinase C from sea urchin eggs

1. Protein kinase C is considered to be ubiquitous in tissues and organs; however, its isolation and characterization have been principally with adult mammalian tissues. 2. There is increasing evidence for the importance of this enzyme during early development. 3. In this study, protein kinase C has been identified and partially characterized in cytosolic fraction from sea urchin eggs. 4. The enzyme was resolved from other protein kinase activities by ion exchange chromatography. 5. Phosphatidylserine and Ca2+ were required for protein kinase C to be active. 6. Diacylglycerol and phorbol ester enhanced the activation of the enzyme.     

The mechanism supplying acetyl-CoA for terpene biosynthesis in sweet potato with black rot: Incorporation of acetate-2-14C, pyruvate-3-14C and citrate-2,4-14C into ipomeamarone

Silica gel thin layer chromatography showed that acetate-2-¹⁴C,pyruvate-3-¹⁴C and citrate-2,4-¹⁴C were incorporated into ipomeamaronein sweet potato root tissues infected by Ceratocystis fimbriata.Rates of incorporation of ¹⁴C, from these 3 substances, intothe CHCl³-CH₃OH-soluble lipid fraction and ipomeamarone wereof the followingder: acetate > pyruvate > citrate ¹This paper constitutes Part 82 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury (Received December 11, 1969; )     

Characterization and identification of glyceryl ether diesters present in tumor cells

The previously unidentified neutral lipid present in tumor tissues has been isolated from Ehrlich ascites cells and unequivocally identified as a lipid class of glyceryl ether diesters containing various degrees of unsaturation, and ranging in approximate molecular weight from 760 to 990. The glyceryl ether diester fraction was shown to be free from neutral plasmalogens (glyceryl diacyl alk-1'-enyl ethers). The tumor lipid was subjected to saponification, transesterification, and lithium aluminum hydride reduction. The glyceryl monoethers that resulted from deacylation were the 1-isomers ranging in hydrocarbon chain length from C(12) to C(24). The predominant glyceryl ethers were the hexadecyl (49%), octadecyl (21%), and octadecenyl (14%) derivatives. Saturated and mono- and polyunsaturated fatty acids ranging in chain length from C(12) to C(24) carbon atoms were esterified to the glyceryl monoether. Gas-liquid chromatography, thin-layer chromatography, and nuclear magnetic resonance and infrared spectroscopy were used to characterize and identify the intact tumor lipid and its derived products.     

Purification and characterization of three distinct glutathione transferases from mouse liver

Three distinct glutathione transferases in the liver cytosol fraction of male NMRI mice have been purified by affinity chromatography and fast protein liquid chromatofocusing. These enzymes account for approximately 95% of the activity detectable with 1-chloro-2,4-dinitrobenzene as electrophilic substrate. Differences between the three forms are manifested in isoelectric points, apparent subunit molecular mass values, amino acid compositions, N-terminal structures, substrate specificities, and sensitivities to inhibitors, as well as in reactions with specific antibodies raised against glutathione transferases from rat and human tissues. The results indicate strongly that the three mouse enzymes are products of different genes. A comparison of the mouse glutathione transferases with rat and human enzymes revealed similarities between the transferases from different species. Mouse glutathione transferases have been named on the basis of their respective subunit compositions.     

Isolation and Identification of α-Ketoaldehydes in Calf and Rabbit Livers

Carbonyl compounds in calf liver stored at 4°C for 5~6 hr after slaughter were investigated, with emphasis on α-ketoaldehydes. After conversion of carbonyls into their 2,4-dinitrophenylhydrazones (2,4-DNPs), they were fractionated by preparative thin-layer chromatography (TLC); at least, twenty-three fractions were separated, among which eighteen fractions contained 2,4-DNPs of α-dicarbonyls. From some of the fractions, crystalline 2,4-DNPs were isolated, and identified by TLC and infrared spectra: pyruvaldehyde, 3-deoxypentosulose, xylosulose, 3-deoxyglucosulose, 2,3-diketogulonic acid or/and dehydroascorbic acid, and formaldehyde were identified, and glucosulose was tentatively identified.

The carbonyl pattern, obtained from rabbit liver frozen in liquid nitrogen immediately after slaughter, was also similar as that from calf liver, and further, the amount of each α-ketoaldehydes remained at the almost same level during storage at 4°C for 24 hr. These results indicate that the identified α-ketoaldehydes are constituents or metabolic intermediates in calf and rabbit livers.

The formation mechanisms of the carbonyls in tissues are discussed.     

Translocation and Complex Formation of Picloram and 2,4-D in Wheat Seedlings

Uptake, translocation and metabolism of ¹⁴C-labelled 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) in seedlings of wheat (Triticum aestivum L.) were studied. The uptake of the herbicides through the upper surface of the first leaf was slow but was almost complete after nine days. Picloram was absorbed faster than 2,4-D. Picloram was also translocated into the stem and the untreated leaves to a greater extent than 2,4-D. Only small fractions of the activity were recovered from the roots and from the nutrient solution. Picloram and 2,4-D formed water-soluble conjugates in the tissues. These conjugates were very labile and hydrolyzed under release of the unchanged herbicides. The isotope from 2,4-D was also incorporated in an insoluble fraction, containing cell walls and proteins. Also from this fraction biologically active 2,4-D could be released by hydrolysis. The formation of the complexes was partly prevented by cycloheximide. It is suggested that herbicide detoxification through complex formation is of importance for the relatively low sensitivity of wheat to auxin herbicides.     

S-(1,2-dicarboxyethyl)glutathione and activity for its synthesis in rat tissues

The contents of S-(1,2-dicarboxyethyl)glutathione (DCE-GS) in several tissues of rat were determined by HPLC. The peptide was present at concentrations (nmol/g tissue) of 119 in lens, 71.6 in liver, and 27.4 in heart. It was, however, not detected in spleen, kidney, cerebrum, or cerebellum. In rat liver, DCE-GS was located primarily in the cytosolic fraction. The substrates for the enzymic synthesis of DCE-GS were GSH and L-malate. In rats, the DCE-GS-synthesizing activity was found to be highest in the liver and in the cytosol of rat liver subcellular fractions. The DCE-GS-synthesizing enzyme was partially purified from rat liver cytosolic fraction by ammonium sulfate fractionation, Phenyl Superose chromatography, hydroxyapatite chromatography, and gel filtration. The molecular mass of the enzyme was estimated to be 53 kDa by gel filtration and SDS-PAGE, showing it to be a monomeric protein. The Km values for GSH and L-malate were 2.3 and 4.0 mM at 37 degrees C, respectively. The enzyme did not utilize 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, p-nitrophenyl bromide, trans-4-phenyl-3-buten-2-one, or p-nitrobenzyl chloride, which were substrates for previously characterized glutathione S-transferases. The isolated enzyme preparation showed no fumarase activity, which supported the conclusion that the formation of DCE-GS was not the result of a nonenzymic reaction following the synthesis of fumarate from L-malate by the isolated enzyme. The N-terminal amino acid of this polypeptide was presumably blocked since no sequence was obtained by automatic sequencing after electro-blotting onto a siliconized-glass fiber (SGF) sheet.     

Antibacterial Potential of 2,4-Di-tert-Butylphenol and Calixarene-Based Prodrugs from Thermophilic Bacillus licheniformis Isolated in Algerian Hot Spring

The present investigation reports the isolation, molecular identification and structure elucidation of antibacterial produced by two thermophilic spore-forming bacteria from hot spring (98?°C) of Guelma (Algeria). Morphological, biochemical and physiological characteristics were carried out. The molecular identification by 16S rRNA and 16-23S rRNA ITS-PCR sequencing identified the thermophilic strains as Bacillus licheniformis with 99% of similarity with GenBank accession numbers KX100031 and KX100032. Phenotypic characterization has mentioned several differences between thermophilic isolates and Bacillus licheniformis ATCC 14580. The ability of the thermophilic spore- forming bacteria to produce antibacterial compounds against two multidrug resistance bacteria Pseudomonas aeruginosa (NR_0754828.1) and Staphylococcus aureus (NR_075000.1) in pure and mixed culture was investigated by Radial Diffusion Assay at 55?°C. Structural elucidation of actives compounds was carried out using gas chromatography–mass spectrometry analyses. Antibacterial potency of the thermophilic isolates might be due to the association between two phenolic compounds: 2,4-Di-tert-butyl-phenol as principal active compound and p-tert-butylcalix[4]arene as prodrugs comparing between gas chromatography–mass spectrometry analysis of pure and mixed extract. To the best of our knowledge, this is the first report showing production of p-tert-butylcalix[4]arene and 2,4-Di-tert-butyl-phenol as extremolytes compounds from thermophilic Bacillus licheniformis at 55?°C.     

固相微萃取技术在白蚁表皮碳氢化合物分析中的应用

利用固相微萃取(SPME)技术对白蚁表皮碳氢化合物进行萃取,选出较适合的萃取头,适宜的萃取温度和时间,同时利用GC-MS分析白蚁表皮碳氢化合物的组成成分及含量。结果表明,该白蚁表皮中含有24种碳氢化合物,主要为11,13-二甲基二十六烷烃(33.34%)和2,4,6,-三甲基二十七烷烃(15.75%)。     

Microbial degradation and detoxification of 2,4-dinitrophenol in aerobic and anoxic processes

A bacterial strain was isolated from a river sediment in Buenos Aires, Argentina, owing to its ability to utilize 2,4-dinitrophenol (2,4-DNP) as the sole carbon, nitrogen and energy source. The strain was identified as Rhodococcus opacus on the basis of its 16S rRNA gene sequence. R. opacus degrades aerobically 0.27 and 0.54 mM within 22 and 28 h, respectively, and releases the nitro groups from 2,4-DNP as nitrites. Aerobic biodegradation processes were performed using a 2-l volume microfermentor at with agitation (200 rpm), and were evaluated by spectrophotometry, high performance liquid chromatography (HPLC) and microbial growth. The absence of 2,4-DNP transformation products was also confirmed by gas chromatography mass spectrometry (GC–MS). As the nitrite released during 2,4-DNP degradation is in addition an environmental toxic agent it was removed by denitrification in an anoxic process. Detoxification was assessed by using luminescent bacteria, algae and seeds toxicity tests. Toxicity was not detected after combining both the aerobic and anoxic processes.     

The occurrence of abscisic acid in inhibitors B1 and C from immature fruit of Ceratonia siliqua L. (carob) and in commercial carob syrup

Summary The presence of abscisic acid in the inhibitors B₁ and C from immature carob fruit, whole and minus seed, has been established by thin-layer and gas chromatography and by combined gas chromatography-mass spectrometry. Abscisic acid has been identified in commercial carob syrup by the same means. Most, if not all, of the growth inhibitory activity in these fractions is accounted for as abscisic acid by quantitative gas chromatography as the methyl ester. Trimethylsilylation of abscisic acid with bis (trimethylsilyl) acetamide in pyridine gives two isomeric tris(trimethylsilyl) derivatives.     

Phytoceramide in Vertebrate Tissues: One Step Chromatography Separation for Molecular Characterization of Ceramide Species

Ceramide is a precursor for complex sphingolipids in vertebrates, while plants contain phytoceramide. By using a novel chromatography purification method we show that phytoceramide comprises a significant proportion of animal sphingolipids. Total ceramide including phytoceramide from mouse tissue (brain, heart, liver) lipid extracts and cell culture (mouse primary astrocytes, human oligodendroglioma cells) was eluted as a single homogenous fraction, and then analyzed by thin layer chromatography, and further characterized by gas chromatography-mass spectrometry (GC-MS). We detected a unique band that migrated between non-hydroxy fatty acyl ceramide and hydroxy fatty acyl ceramide, and identified it as phytoceramide. Using RT-PCR, we confirmed that mouse tissues expressed desaturase 2, an enzyme that has been reported to generate phytoceramide from dihydroceramide. Previously, only trace amounts of phytoceramide were reported in vertebrate intestine, kidney, and skin. While its function is still elusive, this is the first report of phytoceramide characterization in glial cells and vertebrate brain, heart, and liver.     

Demonstration of glycoprotein antigens associated with stomach tumors

After precipitation of the glycoproteins from gastric tumor tissues by caprylic acid, immunochemical methods were applied to the research of specific gastric tumor antigens; this precipitation process by caprylic acid was less aggressive than the use of urea or proteases. The purification of a fraction associated with tumor tissue has been carried out by affinity chromatography with a specific antiserum immobilized on Sepharose. The obtained fraction contains 4 proteic antigens. One of them is common with all the gastric extracts. Neither CEA, nor alpha-foetoprotein has been detected in this fraction. The corresponding antiserum seems to show a tumor specificity, whereas tissue specificity has to be demonstrated.